UBIQUITINATION AND STABILITY OF P53 AND P73

p53的缺失或失活是癌症发展的重要一步。因此，确定p53的水平和活性是如何正常调节的，以及这种调节在癌症中是如何改变的，是很重要的。P53的降解至少部分是通过泛素蛋白水解系统发生的。我们的实验室可以很容易地在表达野生型p53的细胞和转染p53表达dna的细胞中检测到泛素：p53缀合物。这种能力为我们提供了一个很好的系统来研究p53泛素化对不同刺激的反应。初步研究表明，p53泛素化在休眠释放和DNA损伤反应过程中受到调节。我们将确定观察到p53泛素化水平高或低的条件。然后我们将确定高水平的p53泛素化是否与降低的蛋白质半衰期相关。最后，我们将评估MDM2在介导p53泛素化中的潜在作用。这些研究将在正常细胞和癌细胞中进行，以确定p53泛素化的调节是否在癌症中发生改变。已经建立了一种检测系统，其中野生型p53与MDM2共表达时，在转染细胞中有效地泛素化。将对p53进行缺失分析和诱变，以确定p53内泛素化所需的决定因子。癌症衍生的p53突变体对泛素化的易感性不同。我们将确定这种变异的分子基础。此外，两个MDM2突变体将被检查，以解决MDM2促进p53泛素化的机制。最近对p73和p63的克隆揭示了一个可能执行冗余功能的p53相关蛋白家族。我们希望确定这些新发现的p53家族成员的水平和稳定性是否通过调节p53的相同途径受到调节。初步研究表明，某些癌症来源的p53突变体可以促进p73的快速降解。我们将详细阐述这种效应的分子基础。此外，我们将确定促进p73降解的能力是否与突变型p53的功能获得表型相关。

Loss or inactivation of p53 is an important step in cancer development. It is therefore important to determine how the levels and activity of p53 are normally regulated, and how this regulation is altered in cancer. p53 degradation occurs, at least in part, through the ubiquitin-proteolysis system. Our laboratory can readily detect ubiquitin: p53 conjugates in cells that express wild-type p53, and in cells transfected with p53 expression DNAs. This ability affords us an excellent system to study p53 ubiquitination in response to diverse stimuli. Preliminary studies indicate that p53 ubiquitination is regulated during release from quiescence and in response to DNA damage. We will determine conditions when high or low levels of p53 ubiquitination are observed. We will then determine whether high levels of p53 ubiquitination are correlated with a decreased protein half-life. Finally, the potential role of MDM2 in mediating p53 ubiquitination will be assessed. These studies will be done in normal cells and in cancer cells to determine whether the regulation of p53 ubiquitination is altered in cancer. An assay system has been established in which wild-type p53 is efficiently ubiquitinated in transfected cells when coexpressed with MDM2. Deletion analysis and mutagenesis of p53 will be done to define the determinants within p53 required for ubiquitination. Cancer-derived p53 mutants vary in their susceptibility to ubiquitination. We will determine the molecular basis for this variation. In addition, two MDM2 mutants will be examined to address the mechanism by which MDM2 promotes p53 ubiquitination. The recent cloning of p73 and p63 has revealed a family of p53-related proteins that may carry out redundant functions. We wish to determine whether the levels and stability of these newly identified p53 family members are regulated through the same pathways that regulate p53. Preliminary studies indicate that certain cancer-derived p53 mutants can promote the rapid degradation of p73. We will elaborate the molecular basis of this effect. In addition, we will determine whether the ability to promote p73 degradation is associated with a gain-of-function phenotype for mutant p53.